Protein Amyloid Cofactors: Charged Side-Chain Arrays Meet Their Match?

Recent advances in high-resolution structural studies of protein amyloids have revealed parallel in-register cross-ß-sheets with periodic arrays of closely spaced identical residues. What do these structures tell us about the mechanisms of action of common amyloid-promoting factors, such as heparan sulfate (HS), nucleic acids, polyphosphates, anionic phospholipids, and acidic pH?

More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk.

Arthropods face several key challenges in processing concentrated feedstocks of proteins (silk dope) into solid, semi-crystalline silk fibres. Strikingly, independently evolved lineages of silk-producing organisms have converged on the use of liquid crystal intermediates (mesophases) to reduce the viscosity of silk dope and assist the formation of supramolecular structure. However, the exact nature of the liquid-crystal-forming-units (mesogens) in silk dope, and the relationship between liquid crystallinity, protein structure and silk processing is yet to be fully elucidated. In this review, we focus on emerging differences in this area between the canonical silks containing extended-ß-sheets made by silkworms and spiders, and 'non-canonical' silks made by other insect taxa in which the final crystallites are coiled-coils, collagen helices or cross-ß-sheets. We compared the amino acid sequences and processing of natural, regenerated and recombinant silk proteins, finding that canonical and non-canonical silk proteins show marked differences in length, architecture, amino acid content and protein folding. Canonical silk proteins are long, flexible in solution and amphipathic; these features allow them both to form large, micelle-like mesogens in solution, and to transition to a crystallite-containing form due to mechanical deformation near the liquid-solid transition. By contrast, non-canonical silk proteins are short and have rod or lath-like structures that are well suited to act both as mesogens and as crystallites without a major intervening phase transition. Given many non-canonical silk proteins can be produced at high yield in E. coli, and that mesophase formation is a versatile way to direct numerous kinds of supramolecular structure, further elucidation of the natural processing of non-canonical silk proteins may to lead to new developments in the production of advanced protein materials.

Therapeutic approaches in proteinopathies.

A family of maladies known as amyloid disorders, proteinopathy, or amyloidosis, are characterized by the accumulation of abnormal protein aggregates containing cross-ß-sheet amyloid fibrils in many organs and tissues. Often, proteins that have been improperly formed or folded make up these fibrils. Nowadays, most treatments for amyloid illness focus on managing symptoms rather than curing or preventing the underlying disease process. However, recent advances in our understanding of the biology of amyloid diseases have led to the development of innovative therapies that target the emergence and accumulation of amyloid fibrils. Examples of these treatments include the use of small compounds, monoclonal antibodies, gene therapy, and others. In the end, even if the majority of therapies for amyloid diseases are symptomatic, greater research into the biology behind these disorders is identifying new targets for potential therapy and paving the way for the development of more effective treatments in the future.

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-ß Fibrils to Design Adjuvanted Nanovaccines.

Protein fibrils characterized with a cross-ß-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling ß-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I10) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus. The highly conserved epitope from the ectodomain of the matrix protein 2 (M2e) was elongated at the N-terminus of I10 by solid phase peptide synthesis. The chimeric M2e-I10 peptide readily self-assembled into unbranched, long, and twisted fibrils with a diameter between five and eight nm. These cross-ß nanoassemblies were cytocompatible and activated the heterodimeric Toll-like receptor (TLR) 2/6. Upon mice subcutaneous immunization, M2e-fibrils triggered a robust anti-M2e specific immune response, which was dependent on self-assembly and did not require the use of an adjuvant. Overall, this study describes the efficacy of cross-ß fibrils to activate the TLR 2/6 and to stimulate the epitope-specific immune response, supporting usage of these proteinaceous assemblies as a self-adjuvanted delivery system for antigens.

The other prey-capture silk: Fibres made by glow-worms (Diptera: Keroplatidae) comprise cross-ß-sheet crystallites in an abundant amorphous fraction.

Glow-worms (larvae of dipteran genus Arachnocampa) are restricted to moist habitats where they capture flying prey using snares composed of highly extensible silk fibres and sticky mucus droplets. Little is known about the composition or structure of glow-worm snares, or the extent of possible convergence between glow-worm and arachnid capture silks. We characterised Arachnocampa richardsae silk and mucus using X-ray scattering, Fourier transform infrared spectroscopy and amino acid analysis. Silk but not mucus contained crystallites of the cross-ß-sheet type, which occur in unrelated insect silks but have not been reported previously in fibres used for prey capture. Mucus proteins were rich in Gly (28.5%) and existed in predominantly a random coil structure, typical of many adhesive proteins. In contrast, the silk fibres were unusually rich in charged and polar residues, particularly Lys (18.1%), which we propose is related to their use in a highly hydrated state. Comparison of X-ray scattering, infrared spectroscopy and amino acid analysis data suggests that silk fibres contain a high fraction of disordered protein. We suggest that in the native hydrated state, silk fibres are capable of extension via deformation of both disordered regions and cross-ß-sheet crystallites, and that high extensibility is an adaptation promoting successful prey capture. This study illustrates the rich variety of protein motifs that are available for recruitment into biopolymers, and how convergently evolved materials can nevertheless be based on fundamentally different protein structures.